Inverted microscopes are characterised in that light is passed from above through the object to be examined in a transmitted light process and in that the objectives are attached below the object stage. In the case of inverted reflected light microscopes, both the illumination and the observation are carried out through the objective from below. Reflected light microscopes of this type play a central part in mineralogy and metallurgy, whereas inverted transmitted light microscopes are frequently used for the examination or for the handling of biological samples. One advantage of inverted microscopes is that the object to be examined is more accessible, as the imaging optics are for the most part arranged below the sample stage, e.g., in the stand.
Biological samples and samples of low thickness can appear almost transparent in normal microscopic examination. Objects of this type usually have differing optical thicknesses, whereas the light amplitude is not attenuated or is attenuated homogeneously via the sample. The optical path differences which exist when light passes through a sample of this type (phase object) can be rendered visible to the human eye by various contrasting processes. Of the known contrasting processes, such as phase contrast, Hoffmann modulation contrast, relief contrast, VAREL contrast or interference contrast, phase contrast and modulation contrast will be described briefly by way of example in the present document as common representatives of the contrasting processes.
In phase contrast, a ring diaphragm positioned upstream in the illumination beam path is imaged to infinity by a condenser. The illumination beams which pass through the ring diaphragm and penetrate the sample undiffracted (“zero-order diffraction arrangement”) strike in the focal plane (general pupil) of the objective and a phase ring, an annular layer which is adapted by vapour deposition, in such a way that compared to the beams which penetrate the phase plate adjacent to this layer, a phase difference of λ/4 is achieved. In the case of amplitude objects, the diffracted light has with respect to the zero order a phase displacement of 180° (λ/2) and in the case of phase objects, the phase displacement is 90° (λ/4). An additional 90° of displacement in the phase ring produces a total of 180° of displacement, the same phase conditions as in the case of an amplitude object. As a result of additional attenuation of the amplitude in the phase ring, the zero-order intensity is adapted to the orders of diffraction. In the intermediate image plane of the microscope, interference of the orders of diffraction produces an image comparable to an amplitude image. Details having a refractive index higher than the surrounding environment appear darker in this image. The phase ring may be customized to the ring diaphragm in such a way that the ring diaphragm is mapped onto the phase ring. The phase ring is located in the objective pupil which is generally positioned within the objective itself. For phase contrast, use is therefore often made of special objectives in which a phase ring is integrated (for example, by vapour deposition onto a lens).
A combination of dielectric and metallic layers is generally used for constructing a phase contrast layer. The dielectric layers (for example, silicon oxide) serve to adjust the phase shift, while the metallic layers (for example, chromium) serve to adjust the desired degree of transmission.
The selection of the suitable phase ring (and thus of the associated ring diaphragm) is dependent not only on the objective, but also on the sample to be examined, which is characterised by the respective degree of transmission and the respective phase displacement. Furthermore, the size of the phase ring must be configured in accordance with the desired resolution or in accordance with the desired contrast. Special objectives having an integrated phase ring cannot respond flexibly to such differing requirements. That is to say, a flexible response would require the provision of a large number of special objectives, and this increases both complexity and costs. In practice, conventional special objectives are therefore universally usable standard solutions which in many cases cannot provide the desired result for special cases.
In modulation contrast, a pupil plane of the observation beam path contains an arranged plate having strip-like regions of differing transparency (generally 0%, 20% and 100%). In this case, the diffraction image is not acted on symmetrically to the optical axis of the objective. The phase objects which are rendered visible by a microscope of this type display a relief effect similar to that which occurs when an object is illuminated obliquely on one side. On the illumination side, at least one slotted diaphragm is linked conjugately to the modulators in the imaging beam path. The slotted diaphragm is generally mapped onto the mean transmission strip of the imaging-side modulator. Usually, these slotted diaphragms are located within a condenser disc, a specific illumination slot being provided for each magnification.
A device for selectively implementing phase contrast and relief contrast in microscopes is described in EP 0 069 263. Starting from the problem that the modulators required for phase and relief contrast are arranged in the objective pupil and therefore in the usually inaccessible interior of an objective, the aforementioned contrasting processes are generally incompatible with each other. EP 0 069 263 proposes a modulator in the objective pupil that is uniform for both contrasting processes. The transition between the contrasting processes is carried out by changing the diaphragm in the illumination beam path. The modulator in the objective pupil consists of a plate which is only partially transparent and has two concentric annular layers which influence the amplitude or phase of the light. For phase contrast observation, an annular diaphragm is introduced into the illumination beam path before the condenser. Additionally, the annular transparent region of the condenser and objective are mapped onto the phase ring of the modulator located in the objective pupil. For observation in relief contrast, a different diaphragm having a transparent annular segment-shaped region which is mapped onto the other ring is introduced into the illumination beam path, influencing only the amplitude, of the modulator in the objective pupil. The one-sided oblique angle at which the light penetrates the object plane gives rise to a relief effect which makes the object appear three-dimensional. This proposed device accordingly allows a transition between phase contrast and relief contrast without acting on the objective pupil, which is difficult to access, and without exchanging objectives made specifically for the respective contrasting process, merely by changing the diaphragms in the illumination beam path.
This aforementioned solution nevertheless requires a special objective which accommodates the aforementioned modulator consisting of a phase ring and an amplitude ring. Should the size of the phase ring be altered or a different contrasting process be used or should other objectives be utilised, the solution also displays the limited flexibility described at the outset.
A microscope for examining amplitude objects and/or phase objects is described in DE-42 36 803 C2. For this purpose, a circular sector diaphragm having a transparent circular sector, the tip of which is located in the optical axis, is attached in the illumination beam path before the condenser. This circular sector is mapped onto a phase plate, which is attached in the rear focal plane (exit pupil) of the objective, via the condenser and objective. At the location of this imaging, the phase plate has a correspondingly configured phase segment, the tip of which is also located in the optical axis, which displaces the phase of the passing light by λ/4. As a result, this contrasting process combines relief contrast and phase contrast. The flexibility of this solution is therefore also limited.
Finally, a microscope comprising a device for achieving phase contrast is described in CH-294755 which proposes generating an intermediate image of the exit pupil of the objective by means of auxiliary optics in order to introduce a phase plate at this location. Expensive special objectives containing a built-in phase plate may thus be dispensed with. The auxiliary optics proposed is in this case a magnification telescopic lens system which generates an intermediate image of the objective exit pupil (referred to hereinafter as the intermediate pupil). The phase plate is fixed at the location of the intermediate pupil. If use is made of various objectives with differing positions of the intermediate pupils, the telescopic system may be displaced along the optical axis for focusing the image of the ring diaphragm opening onto the phase plate. In the case of normal, upright microscopes, it is according to CH-294755 beneficial to arrange the aforementioned auxiliary optics and the phase plate in such a way that they are able to move individually or together. For dished (inverted) microscopes, the phase plate is to be arranged displaceably.
On account of the need, in the case of the solution of CH-294755, to have to monitor both centering and focusing by means of auxiliary optics in the event of any alteration of the position of the pupil when changing the objective, this approach is impractical.